Chronic intermittent ethanol (CIE) exposure produces longer-lasting molecular adaptations including up-regulation of NR2B gene expression and persistence, which is viewed as an important neurobiological basis for the development of ethanol dependence. However, the exact mechanisms underlying this long lasting phenomenon are still unclear. DNA methylation is one of many epigenetic modifications that can alter gene expression, thus, it represents as an ideal candidate mechanism for CIE-induced long-term effects. Therefore, we hypothesize that CIE-induced DNA demethylation of specific CpG sites in the NR2B gene 5'region may increase accessibility of transcription factors to DNA in 5'region of NR2B gene. Repeated ethanol exposures may promote intracellular signaling mechanisms, which regulate DNA methylation and lead to the long lasting plastic changes in NR2B gene expression. These may contribute to the development of alcohol dependence. To address this hypothesis, we plan to use an existing primary cortical neuronal culture CIE model in our laboratory combining with an animal CIE model to a) examine CIE-induced changes in DNA methylation state by mapping individual CpG dinucleotide of the NR2B gene 5'region, which are responsive to long lasting up-regulation of NR2B gene transcription by using bisulfite genomic sequencing, and also examine the correlation of this changes to the development of ethanol-dependence in mice CIE model;b) determine how CIE-induced changes in methylation state of specific CpG sites near the CREB and AP-1 binding sites may impact transcription factors CREB and AP-1 binding complex formation via examining the alterations in CREB/AP-1-DNA binding affinity by using in vitro methylation, transfection, EMSA, site-direct mutation and ChIP;and c) identify the role of relative signaling pathways mediating CIE to DNA methyltransferase activity as well as NR2B gene transcription, including traditional signaling pathways cAMP/PKA and MAPK/ERK and novel signaling proteins involving in modifications of DNA methylation and persistent expression of the NR2B gene by using specific inhibitors, western blot, ELISA and 2-D gel analysis. The results from this proposal will provide new and valuable insight of molecular mechanisms of ethanol regulating NR2B gene expression through epigenetic modifications, which are expected to serve as a basis for possible intervention of alcohol dependence and alcohol withdrawal syndrome.